This competing renewal application represents a major extension of findings obtained during the previous tenure of this R01 which is in its 26th contiguous year of funding. During this time we identified a number of novel signaling pathways in the functional hyperemic response to neuronal metabolic activity. We have further identified the role played by astrocytes in supplying blood flow to neurons when cortical activity increases. Basically, astrocytes impinge on muscle cells of the microcirculation to activate K+ channels, hyperpolarizing the cell, imposing dilation and increasing blood flow to those neurons which have, or are in the process of developing an oxygen debt. This grant will further investigate new findings that 20-HETE is made and released by astrocytes via cytochrome P450 (CYP) 4A2 activity. The role of astrocytes may be to modify the extent of hyperemic flow by constricting cerebral arterioles which are activated during functional hyperemia, or play an, as of yet, unidentified role. Furthermore, we have identified a diurnal/circadian rhythm in CYP 2C11, 4X1, and 4A2 message indicating that CYP 2nd messengers are controlled or participate in a physiologic mechanism which varies with respect to time of day. These new findings may have important physiologic significance in that certain cerebral vascular pathologies occur at specific times of the day. For example, stroke has been epidemiologically identified to occur in early/mid morning hrs. At present we do not know if these genes are regulated by any of the major clock genes, or if such rhythm is tissue specific. If EET production is regulated at a time out of phase with production of 20-HETE, there may be a diurnal mechanism matching functional hyperemia and autoregulation of cerebral blood flow. Finally, we will study signaling pathways between the primary cell types forming the neurovascular unit (NVU) to determine the functional interplay of 2nd messengers between neurons, astrocytes and the cerebral microcirculation. These studies represent a blending of new and old approaches in understanding the cell types integrating neuronal, astrocytes and micro vascular function with respect to metabolic and myogenic activity in the brain. PUBLIC HEALTH RELEVANCE: The brain is a very active organ which during normal functioning consumes great quantities of oxygen and nutrients - all delivered by blood flow through arteries. When specific regions (i.e., neurons) of the brain are very active, it is advantageous for the brain to be able to increase blood flow to those neurons. We believe the ability to direct blood flow to specific brain regions is accomplished by the signaling of neuron activity levels to the blood vessels serving that region through interconnected cells called astrocytes. We believe that astrocytes detect neuronal activity, and then send signaling molecules to the adjacent vasculature to increase blood flow, and thereby, increase the delivery of oxygen and nutrients. The detection of neuronal activity levels by astrocytes, how astrocytes communicate this increase in demand to the adjacent blood vessels, and how astroyctes and cells of the blood vessel control blood flow, will be determined. This knowledge will aid us in understanding, and hopefully preventing, a major cause of stroke.